A clinical trial in dogs with spontaneous osteosarcoma was performed to assess a recombinant Listeria expressing a chimeric human HER2 (ADXS31-164) as an adjunctive vaccine strategy to prevent metastatic disease and determine immunological correlates of clinical outcome. A total of 118 dogs with appendicular osteosarcoma were recruited into a 1-arm, multicenter, prospective trial of standard of care (SOC) therapy followed by ADXS31-164. ADXS31-164 was well tolerated, with mostly transient, low-grade side effects. Significant differences in median disease-free interval (DFI) or median overall survival (OS) of immunized dogs compared to a historical cohort of dogs receiving SOC only were not observed. Elite survivors (DFI >490 days) showed transient increases in temperature and serum cytokines, including IL-6 and TNF-α, after the first immunization compared to short-term survivors (DFI 150-235 days). However, repeat immunizations in short-term survivors led to improved and comparable pyrexic and cytokine responses to elite survivors. PBMC transcriptomic analysis following vaccinations revealed robust cytotoxic activity in elite but not short-term survivors. Although ADXS31-164 did not significantly extend DFI or OS, immune responses to ADXS31-164 distinguished elite from short-term survivors. Improvement of immune responses over sequential ADXS31-164 administrations supports a future trial design of recurrent immunizations to improve outcomes of otherwise short-term survivors.